Polypeptides capable of inhibiting the binding between leptin and Neuropilin-1

ABSTRACT

The present invention relates to agents capable of inhibiting the binding between Leptin and Neuropilin-1 (NRP1) and uses thereof in the therapeutic field.

FIELD OF THE INVENTION

The present invention relates to agents capable of inhibiting thebinding between Leptin and Neuropilin-1 (NRP1) and uses thereof in thetherapeutic field.

BACKGROUND OF THE INVENTION

Cytokines, hormones and interactions with their cognate receptors playcritical roles in the regulation and homeostasis of variousphysiological systems. Their dysregulation may lead to diseases relatedto metabolism, cancer development, and immune or hematopoiesisdisorders.

Interactions between hormones, cytokines and their cognate receptors arecomplex and may require the implication of various co-receptors thatmodulate in a specific cell type their affinity and the signaltransduction. Therefore, depending on the co-receptors involved,hormones/cytokines may compete with each other and may transduce eitherpositive or negative signals for cell growth, survival and specificfunctions.

Leptin (Lep/OB) is a cytokine-like hormone mainly produced by adipocytesand plays a crucial role in the maintenance of energy balance throughits effect in reducing food intake and increasing energy expenditure. Itactivates receptors (OBR) highly expressed in the brain hypothalamicarcuate nucleus (ARC), a site well known to control body weight (HeikeMünzberg and Christopher D Morrison, Metabolism 2014). Hence, defect inleptin receptor signaling leads to severe obesity. Currently, twoisoforms of the leptin receptors (OBR) are mainly studied, the shortisoform OBRa and the long isoform OBRb.

Leptin mediates most of its effect by activating the long receptorisoform (OBRb), which is the dominant signaling species highly expressedin the brain hypothalamic arcuate nucleus (ARC) (Heike Münzberg andChristopher D Morrison, Metabolism 2014).

However, recent evidences showed that OBR is also expressed inperipheral tissues and that leptin is a more pleiotropic hormone playinga role in other pathophysiological processes including immuneregulation, hematopoiesis, cancer development, and metabolism associateddisorders including type 2 diabetes, kidney function failure andmetabolism associated and age related ocular diseases.

Studies from our group, deciphering the role of adipocytes on thehematopoiesis regulation showed that neuropilin-1 (NRP-1) isoverexpressed in femoral fatty bone marrow (Zakia Belaid et al.Heamatologica 2005). NRP-1 is a transmembrane glycoprotein implicated inaxonal guidance and angiogenesis through specific ligands andco-receptors SEMA/Plexin and VEGF/VEGFR, respectively (Fujisawa H et al,J Neurobiol 2004 and Soker S et al, Cell 1998). NRP-1 has been shown byour group to be involved in 1) the immune response by participating inthe formation of the immune synapse between dendritic cells andT-lymphocytes (R. Tordjman, Yves Lepelletier et al., Nature Immunology2002), 2) the HTLV1 entry in lymphocytes (David Ghez, Yves Lepelletieret al., Journal of Virology 2005) and 3) the regulation of hematopoiesisby blocking granulopoiesis through the inhibition of the production ofgranulocyte colony stimulating factor (G-CSF) by macrophages under theinfluence of adipocytes (Zakia Belaid-Choucair et al. Stem Cell 2008).The latter function was independent of known NRP-1 ligands but involvedleptin produced by adipocytes (unpublished data).

NRP-1 needs to form a complex with receptors belonging to the plexinfamily, which serve as the signal-transducing element for the axonalrepulsion and collapse of neuronal growth cones after SEMA binding tothe NRP-1/Plexin complex (He and Tessier-Lavigne, Cell 1997). NRP-1 mayalso interact with VEGF receptors (VEGF-R) forming a complex, which canbe activated by VEGF165 for normal developmental angiogenesis (Soker Set al. Cell 1998). Based on both the literature and our results ingranulopoiesis regulation, We postulated that the leptin receptor OBRmay form a complex with NRP-1. Interestingly, macrophages involved ingranulopoiesis inhibition expressed both NRP-1 and OBR at their cellsurfaces. NRP-1 and OBR interaction in macrophages was detected bywestern blotting after NRP-1 co-immunoprecipitation.

By using a well described MDA-MB231 (NRP-1 positive and OBR positive)and T47D (OBR low and NRP-1 negative) breast cancer cell linestransduced either by shNRP-1 or cDNA encoding for NRP-1 we have shownthat 1) NRP-1 forms a complex with OBR 2) NRP-1/OBR complex formation isleptin dependent 3) the NRP-1/OBR complex translocates to the nucleus 4)both complex formation and nuclear translocation are dependent on NRP-1and JAK2 phosphorylation by the Serine/Threonine casein kinase2 (CK2).This finding was confirmed by the inhibition of CK2 by 3 differentchemical compounds (TBB, DRB and CX4945) and by RNA silencing thatprevented not only NRP-1 and JAK2 phosphorylation but also the formationof the NRP-1/OBR complex.

Leptin has been reported to regulate more than 64 genes including thosefor growth, cell cycle regulators, extracellular matrix proteins andgene associated with metastasis (Perera C. N et al. J of Endocrinol 2008and EBM 2008).

Besides a major role in energy homeostasis, the adipokine hormone-like“Leptin” is emerging as a pleiotropic cytokine with pro-angiogenicactivity by inducing VEGF and VEGFR2 expression. Multiple signalingmolecules modulate the complex interplay between the vascular system andthe adipocytes (Yihai Cao, Cell Metabolism 2013).

In breast cancer, leptin is considered as a pro-angiogenic marker (RubenRene Gonzalez-Perez et al, Cancers 2013). Anti-VEGF (bevacizumab)treatment of patients with rectal cancer was significantly associatedwith distant metastasis at three years, which correlated with theup-regulation of NRP-1 and activation of inflammatory pathways (Xu Leiet al, cancer Research 2009).

Interestingly, our functional characterization of NRP-1/OBR complexusing breast cancer xenograft model led us to demonstrate that NRP-1modulates leptin function by decreasing cell proliferation, inducingcell migration and lymph node infiltration. Since leptin has beenreported to be pro-angiogenic, we postulated that physiologically, VEGF(a target of Bevacizumab) might play a negative feedback on leptinaction during angiogenesis. During treatment with Bevacizumab, VEGFmight be unable to reduce leptin action and thus NRP-1 overexpressionwill be in the favor of NRP-1/OBR and leptin interactions. This complexwould lead to a non-canonical signaling involved in metastasis and thusdecrease of the overall survival.

As hypothesized, preventing the binding of VEGF to VEGFR by treatment ofMDA-MB231 breast cancer cell line with Avastin alone induces in vitrocell migration, as did leptin alone. This effect was further increasedwhen the cells were co-treated with both Avastin and leptin. Theincrease of leptin effect on cell migration by Avastin treatment raisedthe question whether VEGF may have a negative feed-back on NRP-1/OBRsignaling. Interestingly, we could demonstrate that VEGFs bind to OBR.Thus, we can conclude that blocking VEGF with Avastin increases leptinaction and induces metastasis and other known effect of leptin such asinflammation that is also reported during Avastin therapy through anincrease of NRP-1OBR complex formation. Interestingly our hypothesis isclearly demonstrated by the increase of NRP-1/OBR complex detection byPLA-HRP technology in MDA-MB231 breast cancer cell line treated withAvastin compared to the control cell. The experiment was done in thepresence of normal human serum in order to mimic the physiologiccondition during Avastin therapy.

By using a BioLayer Interferometry technology (BLI,http://www.fortebio.com), we were able to demonstrate a directinteraction between recombinant proteins Leptin and NRP-1. In contrastto other NRP-1 ligands such as VEGF, and Sema3A known as competitors,leptin binds directly to NRP-1 but do not compete with VEGF binding.Similarly, VEGF does not prevent leptin binding to NRP-1. Theseobservations suggest that leptin and VEGF have distinct binding domains.

This finding suggests that anti-VEGF therapy should be combined with ananti-leptin therapy in cancer.

This could also implicate disease associated to immune systemdysfunction where leptin has been clearly associated in the diseaseaggravation.

SUMMARY OF THE INVENTION

The present invention relates to agents capable of inhibiting thebinding between leptin and NRP-1 and uses thereof in the therapeuticfield. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION

One object of the present invention relates to agent capable ofinhibiting the binding between leptin and NRP-1.

As used herein, the term “leptin” has its general meaning in the art andrefers to a protein that is secreted by white adipocytes, and whichplays a major role in the regulation of body weight. In particular,leptin functions as part of a signaling pathway that can inhibit foodintake and/or regulate energy expenditure to maintain constancy of theadipose mass. This protein also has several endocrine functions, and isinvolved in the regulation of immune and inflammatory responses,hematopoiesis, angiogenesis and wound healing. An exemplary human aminoacid sequence of leptin is SEQ ID NO:1.

SEQ ID NO: 1 MHWGTLCGFLWLWPYLFYAQAVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGLDFIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISND LENLRDLLHVLAFSKSCHLPWASGLETL DSLGGVLEASGYSTEVVALSRL QGSLQDMLWQLDLSPGC

As used herein, the term “NRP-1” has its general meaning in the art andrefers to the neuropilin-1 receptor. An exemplary human amino acidsequence of NRP-1 is SEQ ID NO:2.

SEQ ID NO: 2 MERGLPLLCAVLALVLAPAGAFRNDKCGDTIKIESPGYLTSPGYPHSYHPSEKCEWLIQAPDPYQRIMINFNPHFDLEDRDCKYDYVEVFDGENENGHFRGKFCGKIAPPPVVSSGPFLFIKFVSDYETHGAGFSIRYEIFKRGPECSQNYTTPSGVIKSPGFPEKYPNSLECTYIVFAPKMSEIILEFESFDLEPDSNPPGGMFCRYDRLEIWDGFPDVGPHIGRYCGQKTPGRIRSSSGILSMVFYTDSAIAKEGFSANYSVLQSSVSEDFKCMEALGMESGEIHSDQITASSQYSTNWSAERSRLNYPENGWTPGEDSYREWIQVDLGLLRFVTAVGTQGAISKETKKKYYVKTYKIDVSSNGEDWITIK EGNKPVLFQGNTNPTDVVVAVFPK PLITRFVRIKPATWETGISMRFEVYGCKITDYPCSGMLGMVSGLISDSQITSSNQGDRNWMPENIRLVTSRSGWALPPAPHSYINEWLQIDLGEEKIVRGIIIQGGKHRENKVFMRKFKIGYSNNGSDWKMIMDDSKRKAKSFEGNNNYDTPELRTFPALSTRFIRIYPERATHGGLGLRMELLGCEVEAPTAGPTTPNGNLVDECDDDQANCHSGTGDDFQLTGGTTVLATEKPTVIDSTIQSEFPTYGFNCEFGWGSHKTFCHWEHDNHVQLKWSVLTSKTGPIQDHTGDGNFIYSQADENQKGKVARLVSPVVYSQNSAHCMTFWYHMSGSHVGTLRVKLRYQKPEEYDQLVWMAIGHQGDHWKEGRVLLHKSLKLYQVIFEGEIGKGNLGGIAVDDISINNHISQEDCAKPADLDKKNPEIKIDETGSTPGYEGEGEGDKNISRKPGNVLKTLDPILITIIAMSALGVLLGAVCGVVLYCACWHNGMSERNLSALENYNFELVDGVKLKKDKLNTQSTYSEA

In some embodiments, the agents is an isolated, a synthetic orrecombinant polypeptide capable of inhibiting the binding between leptinand NRP-1.

In some embodiments, the polypeptide of the present invention comprisesan amino acid sequence having at least 90% of identity with the sequenceof SEQ ID NO:3 (ENLRDLLHVLAFSKSCHLPWASGLETL) or SEQ ID NO:4(EGNKPVLFQGNTNPTDVVVAVFPK).

According to the invention a first amino acid sequence having at least90% of identity with a second amino acid sequence means that the firstsequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identitywith the second amino acid sequence. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar are the two sequences.Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math, 2:482, 1981; Needleman and Wunsch, J.Mol. Biol., 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A., 85:2444, 1988; Higgins and Sharp, Gene, 73:237-244, 1988;Higgins and Sharp, CABIOS, 5:151-153, 1989; Corpet et al. Nuc. AcidsRes., 16:10881-10890, 1988; Huang et al., Comp. Appls Biosci, 8:155-165,1992; and Pearson et al., Meth. Mol. Biol., 24:307-31, 1994). Altschulet al., Nat. Genet, 6:119-129, 1994, presents a detailed considerationof sequence alignment methods and homology calculations. By way ofexample, the alignment tools ALIGN (Myers and Miller, CABIOS 4:11-17,1989) or LFASTA (Pearson and Lipman, 1988) may be used to performsequence comparisons (Internet Program® 1996, W. R. Pearson and theUniversity of Virginia, fasta20u63 version 2.0u63, release date December1996). ALIGN compares entire sequences against one another, while LFASTAcompares regions of local similarity. These alignment tools and theirrespective tutorials are available on the Internet at the NCSA Website,for instance. Alternatively, for comparisons of amino acid sequences ofgreater than about 30 amino acids, the Blast 2 sequences function can beemployed using the default BLOSUM62 matrix set to default parameters,(gap existence cost of 11, and a per residue gap cost of 1). Whenaligning short peptides (fewer than around 30 amino acids), thealignment should be performed using the Blast 2 sequences function,employing the PAM30 matrix set to default parameters (open gap 9,extension gap 1 penalties). The BLAST sequence comparison system isavailable, for instance, from the NCBI web site; see also Altschul etal., J. Mol. Biol., 215:403-410, 1990; Gish. & States, Nature Genet.,3:266-272, 1993; Madden et al. Meth. Enzymol., 266:131-141, 1996;Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997; and Zhang &Madden, Genome Res., 7:649-656, 1997.

In some embodiments, the polypeptide of the present invention comprisesan amino acid sequence having at least 90% of identity with the sequenceof SEQ ID NO:3 comprises at least 27; 28; 29; 30; 31; 32; 33; 34; 35;36; 37; 38; 39; 40 or more amino acids. In some embodiments, thepolypeptide of the present invention comprises an amino acid sequencehaving at least 90% of identity with the sequence of SEQ ID NO:4comprises at least 24; 25; 26; 27; 28; 29; 30; 31; 32; 33; 34; 35; 36;37; 38; 39; 40 or more amino acids. In some embodiments, the polypeptideof the present invention comprises less than 50 amino acids. In someembodiments, the polypeptide of the present invention comprises lessthan 30 amino acids.

The functional properties of the polypeptide of the present invention,i.e. inhibition of the binding between Leptin and NRP-1, could typicallybe assessed in any functional assay as described in EXAMPLE.

In some embodiments, the polypeptide of the present invention is fusedto at least one heterologous polypeptide to form a fusion protein.

In some embodiments, the polypeptide of the present invention is fusedeither directly or via a spacer at its C-terminal end to the N-terminalend of the heterologous polypeptide, or at its N-terminal end to theC-terminal end of the heterologous polypeptide. As used herein, the term“directly” means that the (first or last) amino acid at the terminal end(N or C-terminal end) of the polypeptide of the present invention isfused to the (first or last) amino acid at the terminal end (N orC-terminal end) of the heterologous polypeptide. In other words, in thisembodiment, the last amino acid of the C-terminal end of saidpolypeptide is directly linked by a covalent bond to the first aminoacid of the N-terminal end of said heterologous polypeptide, or thefirst amino acid of the N-terminal end of said polypeptide is directlylinked by a covalent bond to the last amino acid of the C-terminal endof said heterologous polypeptide. As used herein, the term “spacer”refers to a sequence of at least one amino acid that links thepolypeptide of the invention to the heterologous polypeptide. Such aspacer may be useful to prevent steric hindrances. Typically a spacercomprises 2, 3; 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 16; 17; 18;19; or 20 amino acids.

The polypeptides of the present invention are produced by any techniqueknown per se in the art, such as, without limitation, any chemical,biological, genetic or enzymatic technique, either alone or incombination. For instance, knowing the amino acid sequence of thedesired sequence, one skilled in the art can readily produce saidpolypeptides, by standard techniques for production of amino acidsequences. For instance, they can be synthesized using well-known solidphase method, preferably using a commercially available peptidesynthesis apparatus (such as that made by Applied Biosystems, FosterCity, Calif.) and following the manufacturer's instructions.Alternatively, the polypeptides of the present invention can besynthesized by recombinant DNA techniques as is now well-known in theart. For example, these fragments can be obtained as DNA expressionproducts after incorporation of DNA sequences encoding the desired(poly)peptide into expression vectors and introduction of such vectorsinto suitable eukaryotic or prokaryotic hosts that will express thedesired polypeptide, from which they can be later isolated usingwell-known techniques.

Polypeptides or fusion proteins of the invention can be used in anisolated (e.g., purified) form or contained in a vector, such as amembrane or lipid vesicle (e.g. a liposome).

In some embodiments, it is contemplated that polypeptides of the presentinvention may be modified in order to improve their therapeuticefficacy. Such modification of therapeutic compounds may be used todecrease toxicity, increase circulatory time, or modify biodistribution.For example, the toxicity of potentially important therapeutic compoundscan be decreased significantly by combination with a variety of drugcarrier vehicles that modify biodistribution. A strategy for improvingdrug viability is the utilization of water-soluble polymers. Variouswater-soluble polymers have been shown to modify biodistribution,improve the mode of cellular uptake, change the permeability throughphysiological barriers; and modify the rate of clearance from the body.To achieve either a targeting or sustained-release effect, water-solublepolymers have been synthesized that contain drug moieties as terminalgroups, as part of the backbone, or as pendent groups on the polymerchain. For example, Pegylation is a well-established and validatedapproach for the modification of a range of polypeptides (Chapman,2002). The benefits include among others: (a) markedly improvedcirculating half-lives in vivo due to either evasion of renal clearanceas a result of the polymer increasing the apparent size of the moleculeto above the glomerular filtration limit, and/or through evasion ofcellular clearance mechanisms; (b) reduced antigenicity andimmunogenicity of the molecule to which PEG is attached; (c) improvedpharmacokinetics; (d) enhanced proteolytic resistance of the conjugatedprotein (Cunningham-Rundles et. al., 1992); and (e) improved thermal andmechanical stability of the PEGylated polypeptide. Therefore,advantageously, the polypeptides of the invention may be covalentlylinked with one or more polyethylene glycol (PEG) group(s). One skilledin the art can select a suitable molecular mass for PEG, based on howthe pegylated polypeptide will be used therapeutically by consideringdifferent factors including desired dosage, circulation time, resistanceto proteolysis, immunogenicity, etc. In some embodiments, additionalsites for PEGylation can be introduced by site-directed mutagenesis byintroducing one or more lysine residues. For instance, one or morearginine residues may be mutated to a lysine residue. In someembodiments, additional PEGylation sites are chemically introduced bymodifying amino acids on polypeptides of the invention. In someembodiments, PEGs are conjugated to the polypeptides or fusion proteinsthrough a linker. Suitable linkers are well known to the skilled person.

A further object of the present invention relates to a nucleic acidencoding for a polypeptide of the present invention.

As used herein, the term “nucleic acid” has its general meaning in theart and refers to a DNA or RNA molecule. However, the term capturessequences that include any of the known base analogues of DNA and RNAsuch as, but not limited to 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fiuorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, -uracil-5-oxyacetic acid methylester, uracil-5-oxyaceticacid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

In some embodiment, the nucleic acid is included in a suitable vector,such as a plasmid, cosmid, episome, artificial chromosome, phage orviral vector. So, a further object of the present invention relates to avector and an expression cassette in which a nucleic acid encoding forthe polypeptide of the present invention is associated with suitableelements for controlling transcription (in particular promoter, enhancerand, optionally, terminator) and, optionally translation, and also therecombinant vectors into which a nucleic acid in accordance with theinvention is inserted. These recombinant vectors may, for example, becloning vectors, or expression vectors. As used herein, the terms“vector”, “cloning vector” and “expression vector” mean the vehicle bywhich a DNA or RNA sequence (e.g. a foreign gene) can be introduced intoa host cell, so as to transform the host and promote expression (e.g.transcription and translation) of the introduced sequence. Anyexpression vector for animal cell can be used. Examples of suitablevectors include pAGE107 (Miyaji et al., 1990), pAGE103 (Mizukami andItoh, 1987), pHSG274 (Brady et al., 1984), pKCR (O'Hare et al., 1981),pSG1 beta d2-4 (Miyaji et al., 1990) and the like. Other examples ofplasmids include replicating plasmids comprising an origin ofreplication, or integrative plasmids, such as for instance pUC, pcDNA,pBR, and the like. Other examples of viral vectors include adenoviral,retroviral, herpes virus and AAV vectors. Such recombinant viruses maybe produced by techniques known in the art, such as by transfectingpackaging cells or by transient transfection with helper plasmids orviruses. Typical examples of virus packaging cells include PA317 cells,PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols forproducing such replication-defective recombinant viruses may be foundfor instance in WO 95/14785, WO 96/22378, U.S. Pat. Nos. 5,882,877,6,013,516, 4,861,719, 5,278,056 and WO 94/19478. Examples of promotersand enhancers used in the expression vector for animal cell includeearly promoter and enhancer of SV40 (Mizukami and Itoh, 1987), LTRpromoter and enhancer of Moloney mouse leukemia virus (Kuwana et al.,1987), promoter (Mason et al., 1985) and enhancer (Gillies et al., 1983)of immunoglobulin H chain and the like.

A further aspect of the invention relates to a host cell comprising anucleic acid encoding for the polypeptide of the present invention. Inparticular, a subject of the present invention is a prokaryotic oreukaryotic host cell genetically transformed with at least one nucleicacid of the present invention. The term “transformation” means theintroduction of a “foreign” (i.e. extrinsic or extracellular) gene, DNAor RNA sequence to a host cell, so that the host cell will express theintroduced gene or sequence to produce a desired substance, typically aprotein or enzyme coded by the introduced gene or sequence. A host cellthat receives and expresses introduced DNA or RNA has been“transformed”. In some embodiments, for expressing and producingpolypeptides or fusion proteins of the invention, prokaryotic cells, inparticular E. coli cells will be chosen. Actually, according to theinvention, it is not mandatory to produce the polypeptide or the fusionprotein of the invention in a eukaryotic context that will favorpost-translational modifications (e.g. glycosylation). Furthermore,prokaryotic cells have the advantages to produce protein in largeamounts. If a eukaryotic context is needed, yeasts (e.g. saccharomycesstrains) may be particularly suitable since they allow production oflarge amounts of proteins. Otherwise, typical eukaryotic cell lines suchas CHO, BHK-21, COS-7, C127, PER.C6, YB2/0 or HEK293 could be used, fortheir ability to process to the right post-translational modificationsof the fusion protein of the invention. The construction of expressionvectors in accordance with the invention, and the transformation of thehost cells can be carried out using conventional molecular biologytechniques. The polypeptide or the fusion protein of the invention canfor example, be obtained by culturing genetically transformed cells inaccordance with the invention and recovering the polypeptide or thefusion protein expressed by said cell, from the culture. They may then,if necessary, be purified by conventional procedures, known inthemselves to those skilled in the art, for example by fractionalprecipitation, in particular ammonium sulfate precipitation,electrophoresis, gel filtration, affinity chromatography, etc. Inparticular, conventional methods for preparing and purifying recombinantproteins may be used for producing the proteins in accordance with theinvention.

The present invention also related to an antibody or an aptamer, whichspecifically binds to a polypeptide of the present invention.

As used herein, the term “antibody” is thus used to refer to anyantibody-like molecule that has an antigen binding region, and this termincludes antibody fragments that comprise an antigen binding domain suchas Fab′, Fab, F(ab′)2, single domain antibodies (DABs), TandAbs dimer,Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies,minibodies, diabodies, bispecific antibody fragments, bibody, tribody(scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody;kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager,scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domainantibody, bispecific format); SIP (small immunoprotein, a kind ofminibody); SMIP (“small modular immunopharmaceutical” scFv-Fc dimer;DART (ds-stabilized diabody “Dual Affinity ReTargeting”); small antibodymimetics comprising one or more CDRs and the like. The techniques forpreparing and using various antibody-based constructs and fragments arewell known in the art (see Kabat et al., 1991, specifically incorporatedherein by reference). Diabodies, in particular, are further described inEP 404, 097 and WO 93/1 1 161; whereas linear antibodies are furtherdescribed in Zapata et al. (1995). Antibodies can be fragmented usingconventional techniques. For example, F(ab′)2 fragments can be generatedby treating the antibody with pepsin. The resulting F(ab′)2 fragment canbe treated to reduce disulfide bridges to produce Fab′ fragments. Papaindigestion can lead to the formation of Fab fragments. Fab, Fab′ andF(ab′)2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies,diabodies, bispecific antibody fragments and other fragments can also besynthesized by recombinant techniques or can be chemically synthesized.Techniques for producing antibody fragments are well known and describedin the art. For example, each of Beckman et al., 2006; Holliger &Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001; Reiter et al.,1996; and Young et al., 1995 further describe and enable the productionof effective antibody fragments.

In natural antibodies, two heavy chains are linked to each other bydisulfide bonds and each heavy chain is linked to a light chain by adisulfide bond. There are two types of light chain, lambda (l) and kappa(κ). There are five main heavy chain classes (or isotypes) whichdetermine the functional activity of an antibody molecule: IgM, IgD,IgG, IgA and IgE. Each chain contains distinct sequence domains. Thelight chain includes two domains, a variable domain (VL) and a constantdomain (CL). The heavy chain includes four domains, a variable domain(VH) and three constant domains (CH1, CH2 and CH3, collectively referredto as CH). The variable regions of both light (VL) and heavy (VH) chainsdetermine binding recognition and specificity to the antigen. Theconstant region domains of the light (CL) and heavy (CH) chains conferimportant biological properties such as antibody chain association,secretion, trans-placental mobility, complement binding, and binding toFc receptors (FcR). The Fv fragment is the N-terminal part of the Fabfragment of an immunoglobulin and consists of the variable portions ofone light chain and one heavy chain. The specificity of the antibodyresides in the structural complementarity between the antibody combiningsite and the antigenic determinant. Antibody combining sites are made upof residues that are primarily from the hypervariable or complementaritydetermining regions (CDRs). Occasionally, residues from nonhypervariableor framework regions (FR) influence the overall domain structure andhence the combining site. Complementarity Determining Regions or CDRsrefer to amino acid sequences, which together define the bindingaffinity and specificity of the natural Fv region of a nativeimmunoglobulin binding site. The light and heavy chains of animmunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3and H-CDR1, H-CDR2, H-CDR3, respectively. An antigen-binding site,therefore, includes six CDRs, comprising the CDR set from each of aheavy and a light chain V region. Framework Regions (FRs) refer to aminoacid sequences interposed between CDRs.

The term “Fab” denotes an antibody fragment having a molecular weight ofabout 50,000 and antigen binding activity, in which about a half of theN-terminal side of H chain and the entire L chain, among fragmentsobtained by treating IgG with a protease, papaine, are bound togetherthrough a disulfide bond.

The term “F(ab′)2” refers to an antibody fragment having a molecularweight of about 100,000 and antigen binding activity, which is slightlylarger than the Fab bound via a disulfide bond of the hinge region,among fragments obtained by treating IgG with a protease, pepsin.

The term “Fab′” refers to an antibody fragment having a molecular weightof about 50,000 and antigen binding activity, which is obtained bycutting a disulfide bond of the hinge region of the F(ab′)2.

A single chain Fv (“scFv”) polypeptide is a covalently linked VH:VLheterodimer which is usually expressed from a gene fusion including VHand VL encoding genes linked by a peptide-encoding linker. “dsFv” is aVH::VL heterodimer stabilized by a disulfide bond. Divalent andmultivalent antibody fragments can form either spontaneously byassociation of monovalent scFvs, or can be generated by couplingmonovalent scFvs by a peptide linker, such as divalent sc(Fv)2.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites.

Monoclonal antibodies may be generated using the method of Kohler andMilstein (Nature, 256:495, 1975). To prepare monoclonal antibodiesuseful in the invention, a mouse or other appropriate host animal isimmunized at suitable intervals (e.g., twice-weekly, weekly,twice-monthly or monthly) with the appropriate antigenic forms (i.e.polypeptides of the present invention). The animal may be administered afinal “boost” of antigen within one week of sacrifice. It is oftendesirable to use an immunologic adjuvant during immunization. Suitableimmunologic adjuvants include Freund's complete adjuvant, Freund'sincomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponinadjuvants such as QS21 or Quil A, or CpG-containing immunostimulatoryoligonucleotides. Other suitable adjuvants are well-known in the field.The animals may be immunized by subcutaneous, intra-peritoneal,intramuscular, intravenous, intranasal or other routes. A given animalmay be immunized with multiple forms of the antigen by multiple routes.

Briefly, the recombinant polypeptide of the present invention may beprovided by expression with recombinant cell lines. Recombinant forms ofthe polypeptides may be provided using any previously described method.Following the immunization regimen, lymphocytes are isolated from thespleen, lymph node or other organ of the animal and fused with asuitable myeloma cell line using an agent such as polyethylene glycol toform a hydridoma. Following fusion, cells are placed in media permissivefor growth of hybridomas but not the fusion partners using standardmethods. Following culture of the hybridomas, cell supernatants areanalyzed for the presence of antibodies of the desired specificity,i.e., that selectively bind the antigen. Suitable analytical techniquesinclude ELISA, flow cytometry, immunoprecipitation, and westernblotting. Other screening techniques are well-known in the field.Preferred techniques are those that confirm binding of antibodies toconformationally intact, natively folded antigen, such as non-denaturingELISA, flow cytometry, and immunoprecipitation.

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The Fc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)2 fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated as Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity-determining regions (CDR1 through CDR5). The CDRs, andin particular the CDR5 regions, and more particularly the heavy chainCDR5, are largely responsible for antibody specificity.

It is now well-established in the art that the non CDR regions of amammalian antibody may be replaced with similar regions of conspecificor hetero-specific antibodies while retaining the epitope specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody.

In some embodiments, the antibody is a humanized antibody. As usedherein, “humanized” describes antibodies wherein some, most or all ofthe amino acids outside the CDR regions are replaced with correspondingamino acids derived from human immunoglobulin molecules. Methods ofhumanization include, but are not limited to, those described in U.S.Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and5,859,205, which are hereby incorporated by reference. The above U.S.Pat. Nos. 5,585,089 and 5,693,761, and WO 90/07861 also propose fourpossible criteria which may used in designing the humanized antibodies.The first proposal was that for an acceptor, use a framework from aparticular human immunoglobulin that is unusually homologous to thedonor immunoglobulin to be humanized, or use a consensus framework frommany human antibodies. The second proposal was that if an amino acid inthe framework of the human immunoglobulin is unusual and the donor aminoacid at that position is typical for human sequences, then the donoramino acid rather than the acceptor might be selected. The thirdproposal was that in the positions immediately adjacent to the 3 CDRs inthe humanized immunoglobulin chain, the donor amino acid rather than theacceptor amino acid might be selected. The fourth proposal was to usethe donor amino acid reside at the framework positions at which theamino acid is predicted to have a side chain atom within 3A of the CDRsin a three dimensional model of the antibody and is predicted to becapable of interacting with the CDRs. The above methods are merelyillustrative of some of the methods that one skilled in the art couldemploy to make humanized antibodies. One of ordinary skill in the artwill be familiar with other methods for antibody humanization.

In some embodiments, some, most or all of the amino acids outside theCDR regions have been replaced with amino acids from humanimmunoglobulin molecules but where some, most or all amino acids withinone or more CDR regions are unchanged. Small additions, deletions,insertions, substitutions or modifications of amino acids arepermissible as long as they would not abrogate the ability of theantibody to bind a given antigen. Suitable human immunoglobulinmolecules would include IgG1, IgG2, IgG3, IgG4, IgA and IgM molecules. A“humanized” antibody retains a similar antigenic specificity as theoriginal antibody. However, using certain methods of humanization, theaffinity and/or specificity of binding of the antibody may be increasedusing methods of “directed evolution”, as described by Wu et al.,/Mol.Biol. 294:151, 1999. The contents of which are incorporated herein byreference.

Fully human monoclonal antibodies can also be prepared by immunizingtransgenic mice for large portions of human immunoglobulin heavy andlight chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369,5,545,806, 5,545,807, 6,150,584, and references cited therein, thecontents of which are incorporated herein by reference. These animalshave been genetically modified such that there is a functional deletionin the production of endogenous (e.g., murine) antibodies. The animalsare further modified to contain all or a portion of the human germ-lineimmunoglobulin gene locus such that immunization of these animals willresult in the production of fully human antibodies to the antigen ofinterest. Following immunization of these mice (e.g., XenoMouse(Abgenix), HuMAb mice (Medarex/GenPharm), monoclonal antibodies can beprepared according to standard hybridoma technology. These monoclonalantibodies will have human immunoglobulin amino acid sequences andtherefore will not provoke human anti-mouse antibody (KAMA) responseswhen administered to humans. In vitro methods also exist for producinghuman antibodies. These include phage display technology (U.S. Pat. Nos.5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S.Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents areincorporated herein by reference.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)2, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)2 fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornon-human sequences. The present invention also includes so-calledsingle chain antibodies.

The various antibody molecules and fragments may derive from any of thecommonly known immunoglobulin classes, including but not limited to IgA,secretory IgA, IgE, IgG and IgM. IgG subclasses are also well known tothose in the art and include but are not limited to human IgG1, IgG2,IgG3 and IgG4.

Aptamers are a class of molecule that represents an alternative toantibodies in term of molecular recognition. Aptamers areoligonucleotide sequences with the capacity to recognize virtually anyclass of target molecules with high affinity and specificity. Suchligands may be isolated through Systematic Evolution of Ligands byEXponential enrichment (SELEX) of a random sequence library. The randomsequence library is obtainable by combinatorial chemical synthesis ofDNA. In this library, each member is a linear oligomer, eventuallychemically modified, of a unique sequence. Peptide aptamers consists ofa conformationnaly constrained antibody variable region displayed by aplatform protein, such as E. coli Thioredoxin A that are selected fromcombinatorial libraries by two hybrid methods (Colas et al., 1996).

A further object of the present invention relates to a method oftreating a cancer in a subject in need thereof comprising administeringto the subject a therapeutically effective amount of a polypeptide,nucleic acid, antibody or aptamer of the present invention.

As used herein, the term “treatment” or “treat” refer to bothprophylactic or preventive treatment as well as curative or diseasemodifying treatment, including treatment of patient at risk ofcontracting the disease or suspected to have contracted the disease aswell as patients who are ill or have been diagnosed as suffering from adisease or medical condition, and includes suppression of clinicalrelapse. The treatment may be administered to a subject having a medicaldisorder or who ultimately may acquire the disorder, in order toprevent, cure, delay the onset of, reduce the severity of, or ameliorateone or more symptoms of a disorder or recurring disorder, or in order toprolong the survival of a subject beyond that expected in the absence ofsuch treatment. By “therapeutic regimen” is meant the pattern oftreatment of an illness, e.g., the pattern of dosing used duringtherapy. A therapeutic regimen may include an induction regimen and amaintenance regimen. The phrase “induction regimen” or “inductionperiod” refers to a therapeutic regimen (or the portion of a therapeuticregimen) that is used for the initial treatment of a disease. Thegeneral goal of an induction regimen is to provide a high level of drugto a patient during the initial period of a treatment regimen. Aninduction regimen may employ (in part or in whole) a “loading regimen”,which may include administering a greater dose of the drug than aphysician would employ during a maintenance regimen, administering adrug more frequently than a physician would administer the drug during amaintenance regimen, or both. The phrase “maintenance regimen” or“maintenance period” refers to a therapeutic regimen (or the portion ofa therapeutic regimen) that is used for the maintenance of a patientduring treatment of an illness, e.g., to keep the patient in remissionfor long periods of time (months or years). A maintenance regimen mayemploy continuous therapy (e.g., administering a drug at a regularintervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy(e.g., interrupted treatment, intermittent treatment, treatment atrelapse, or treatment upon achievement of a particular predeterminedcriteria [e.g., disease manifestation, etc.]).

As used herein, the term “cancer” has its general meaning in the art andincludes, but is not limited to, solid tumors and blood-borne tumors Theterm cancer includes diseases of the skin, tissues, organs, bone,cartilage, blood and vessels. The term “cancer” further encompasses bothprimary and metastatic cancers. Examples of cancers that may treated bymethods and compositions of the invention include, but are not limitedto, cancer cells from the bladder, blood, bone, bone marrow, brain,breast, colon, esophagus, gastrointestinal, gum, head, kidney, liver,lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue,or uterus. In addition, the cancer may specifically be of the followinghistological type, though it is not limited to these: neoplasm,malignant; carcinoma; carcinoma, undifferentiated; giant and spindlecell carcinoma; small cell carcinoma; papillary carcinoma; squamous cellcarcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrixcarcinoma; transitional cell carcinoma; papillary transitional cellcarcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposiscoli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolaradenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;papillary and follicular adenocarcinoma; nonencapsulating sclerosingcarcinoma; adrenal cortical carcinoma; endometroid carcinoma; skinappendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma;ceruminous; adenocarcinoma; mucoepidermoid carcinoma;cystadenocarcinoma; papillary cystadenocarcinoma; papillary serouscystadenocarcinoma; mucinous cystadenocarcinoma; mucinousadenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma;medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget'sdisease, mammary; acinar cell carcinoma; adenosquamous carcinoma;adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarianstromal tumor, malignant; thecoma, malignant; granulosa cell tumor,malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydigcell tumor, malignant; lipid cell tumor, malignant; paraganglioma,malignant; extra-mammary paraganglioma, malignant; pheochromocytoma;glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficialspreading melanoma; malign melanoma in giant pigmented nevus;epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma;fibrous histiocytoma, malignant; myxosarcoma; liposarcoma;leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolarrhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerianmixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma;mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor,malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma;embryonal carcinoma; teratoma, malignant; struma ovarii, malignant;choriocarcinoma; mesonephroma, malignant; hemangiosarcoma;hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma,malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma;chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma;giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant;ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblasticfibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant;ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillaryastrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma;malignant lymphoma, small lymphocytic; malignant lymphoma, large cell,diffuse; malignant lymphoma, follicular; mycosis fungoides; otherspecified non-Hodgkin's lymphomas; malignant histiocytosis; multiplemyeloma; mast cell sarcoma; immunoproliferative small intestinaldisease; leukemia; lymphoid leukemia; plasma cell leukemia;erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia;basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mastcell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairycell leukemia.

By a “therapeutically effective amount” is meant a sufficient amount ofpolypeptide, nucleic acid, antibody or aptamer of the present inventionfor reaching a therapeutic effect. It will be understood, however, thatthe total daily usage of the compounds and compositions of the presentinvention will be decided by the attending physician within the scope ofsound medical judgment. The specific therapeutically effective doselevel for any particular subject will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;activity of the specific compound employed; the specific compositionemployed, the age, body weight, general health, sex and diet of thesubject; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts. Forexample, it is well within the skill of the art to start doses of thecompound at levels lower than those required to achieve the desiredtherapeutic effect and to gradually increase the dosage until thedesired effect is achieved. However, the daily dosage of the productsmay be varied over a wide range from 0.01 to 1,000 mg per adult per day.Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0,10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient forthe symptomatic adjustment of the dosage to the subject to be treated. Amedicament typically contains from about 0.01 mg to about 500 mg of theactive ingredient, typically from 1 mg to about 100 mg of the activeingredient. An effective amount of the drug is ordinarily supplied at adosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day,especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

In some embodiments, the polypeptide, nucleic acid, antibody or aptamerof the present invention is used in combination with a chemotherapeuticagent for the treatment of cancer. Chemotherapeutic agents include, butare not limited to alkylating agents such as thiotepa andcyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammall and calicheamicinomegall; dynemicin, including dynemicin A; bisphosphonates, such asclodronate; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores, aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharidecomplex); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonicacid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes(especially T-2 toxin, verracurin A, roridin A and anguidine); urethan;vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide;thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil;gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinumcoordination complexes such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO);retinoids such as retinoic acid; capecitabine; and pharmaceuticallyacceptable salts, acids or derivatives of any of the above.

In some embodiments, the polypeptide, nucleic acid, antibody or aptamerof the present invention is used in combination with antiangiogenicagents. As used herein an “antiangiogensis agents” refers to a moleculethat inhibits VEGF or VEGFR-mediated angiogenesis, vasculogenesis, orundesirable vascular permeability. For example, an anti-VEGF therapeuticmay be an antibody directed against VEGF or VEGFR. An anti-VEGF antibodywill usually not bind to other VEGF homologues such as VEGF-B or VEGF-C,or other growth factors such as P1GF, PDGF or bFGF. A preferredanti-VEGF antibody is a monoclonal antibody that binds to the sameepitope as the monoclonal anti-VEGF antibody A4.6.1 produced byhybridoma ATCC® HB 10709 and is a high-affinity anti-VEGF antibody. A“high-affinity anti-VEGF antibody” has at least 10-fold better affinityfor VEGF than the monoclonal anti-VEGF antibody A4.6.1. Preferably theanti-VEGF antibody is a recombinant humanized anti-VEGF monoclonalantibody fragment generated according to WO 98/45331, including anantibody comprising the CDRs or the variable regions of Y0317. Morepreferably, anti-VEGF antibody is the antibody fragment known asranibizumab (LUCENTIS®). The anti-VEGF antibody ranibizumab is ahumanized, affinity-matured anti-human VEGF Fab fragment. Ranibizumab isproduced by standard recombinant technology methods in E. coliexpression vector and bacterial fermentation. Ranibizumab is notglycosylated and has a molecular mass of −48,000 daltons. See WO98/45331and U.S. 2003/0190317.

Antiangiogenic agents include but are not limited to bevacizumab (rhuMabVEGF, Avastin®, Genentech, South San Francisco Calif.), ranibizumab(rhuFAb V2, Lucentis®, Genentech), pegaptanib (Macugen®, EyetechPharmaceuticals, New York N.Y.), sunitinib maleate (Sutent®, Pfizer,Groton Conn.), ramucirumab (Cyramza®, Eli Lilly and Company,Indianapolis, Ind. 46285, USA US License).

Another object of the present invention relates to a pharmaceuticalcomposition comprising the polypeptide, nucleic acid, antibody oraptamer of the present invention and a pharmaceutically acceptablecarrier. Typically, the polypeptide, nucleic acid, antibody or aptamerof the present invention can be combined with pharmaceuticallyacceptable excipients, and optionally sustained-release matrices, suchas biodegradable polymers, to form therapeutic compositions.“Pharmaceutically” or “pharmaceutically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to a mammal, especially ahuman, as appropriate. A pharmaceutically acceptable carrier orexcipient refers to a non-toxic solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type. Inthe pharmaceutical compositions of the present invention for oral,sublingual, subcutaneous, intramuscular, intravenous, transdermal, localor rectal administration, the active principle, alone or in combinationwith another active principle, can be administered in a unitadministration form, as a mixture with conventional pharmaceuticalsupports, to the subjects. Suitable unit administration forms compriseoral-route forms such as tablets, gel capsules, powders, granules andoral suspensions or solutions, sublingual and buccal administrationforms, aerosols, implants, subcutaneous, transdermal, topical,intraperitoneal, intramuscular, intravenous, sub-dermal, transdermal,intrathecal and intranasal administration forms and rectaladministration forms. Typically, the pharmaceutical compositions containvehicles, which are pharmaceutically acceptable for a formulationcapable of being injected. These may be in particular isotonic, sterile,saline solutions (monosodium or disodium phosphate, sodium, potassium,calcium or magnesium chloride and the like or mixtures of such salts),or dry, especially freeze-dried compositions which upon addition,depending on the case, of sterilized water or physiological saline,permit the constitution of injectable solutions. The pharmaceuticalforms suitable for injectable use include sterile aqueous solutions ordispersions; formulations including sesame oil, peanut oil or aqueouspropylene glycol; and sterile powders for the extemporaneous preparationof sterile injectable solutions or dispersions. In all cases, the formmust be sterile and must be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. Solutions comprisingcompounds of the present invention as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The polypeptide, nucleic acid, antibody or aptamer ofthe present invention can be formulated into a composition in a neutralor salt form. Pharmaceutically acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike. The carrier can also be a solvent or dispersion medium containing,for example, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetables oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminiummonostearate and gelatin. Sterile injectable solutions are prepared byincorporating the active compounds in the required amount in theappropriate solvent with several of the other ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum-drying andfreeze-drying techniques which yield a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof. The preparation of more, or highlyconcentrated solutions for direct injection is also contemplated, wherethe use of DMSO as solvent is envisioned to result in extremely rapidpenetration, delivering high concentrations of the active agents to asmall tumor area. Upon formulation, solutions will be administered in amanner compatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms, such as the type of injectable solutionsdescribed above, but drug release capsules and the like can also beemployed. For parenteral administration in an aqueous solution, forexample, the solution should be suitably buffered if necessary and theliquid diluent first rendered isotonic with sufficient saline orglucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous and intra-peritonealadministration. In this connection, sterile aqueous media, which can beemployed, will be known to those of skill in the art in light of thepresent disclosure. Some variation in dosage will necessarily occurdepending on the condition of the subject being treated. The personresponsible for administration will, in any event, determine theappropriate dose for the individual subject.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIG. 1 shows the OB:NRP-1 interaction with VEGF

FIG. 2 shows the OB:VEGF165:NRP1 docking

FIG. 3 shows the NRP1:OB peptides interaction

FIG. 4 shows the increase of NRP-1/OBR complex formation and MDA—MD231cell migration following Avastin treatment

EXAMPLE

Material & Methods

Bio-Layer Interferometry

Bio-layer interferometry (BLI) is a label-free technique that issensitive to an increase of mass bound to the biosensor enablingprotein-protein interaction characterization.

Ligands preparation: proteins were incubated in a PBS buffer with a 1:3ratio molar ratio of biotin (biotin-PEG4-NHS from Pierce EZ kit,prepared following the manufacturer's instructions). Free biotin wasremoved using a desalting column (Pierce). The biotinylated protein(called ligand) was immobilized onto streptavidin biosensor tips anddipped into wells containing the buffer with the analyte of interest(association) or without (dissociation).

Experimental conditions were as follow: total volume in each well: 200μl; shake speed: 1,000 rpm. For simple protein:protein interactions anassociation phase was followed by a dissociation phase. For competitionexperiments, the association phase was followed by another associationphase with a second analyte instead of a dissociation phase.

Sensorgrams were background corrected, smoothed with the Savitzky-Golayalgorithm and analyzed using OctetRED instrument software (ForteBio DataAnalysis version 7.1). Experimental sensorgrams were first fit to a 1:1model. The 1:1 model was accepted if the Chi² test was below 3 and theR² was above 0.9. When the 1:1 model was rejected, the model with thelowest Chi² and the highest R² was then selected.

Molecular Docking Experiment

Preparation of the Protein Structures

The structure of VEGF (PDB ID: 4DEQ) and Leptin (PDB ID: 1AX8) wereextracted from the protein databank (ref Berman). Since the leptinstructure was mutated in the original PDB (W100E), we reversed themutation to the wild type leptin with PyMol (ref Delano). Hydrogens andpartial charges were added using the dockprep routine from Chimera (refPettersen).

Blind Docking Experiment.

We used a hierarchical blind docking protocol comprising PatchDock webserver (ref Schneidman-Duchovny) for the first step with defaultparameters. The top 1000 solutions from PatchDock were refined andreranked using Firedock server (ref Schneidman-duchovny 2). The top 10reranked solutions were optimized with RosettaDock as implemented inROSIE (ref Lyskov) with the no-refine parameter. Consensus binding mode,illustrated in FIG. 2 was extracted from the top solutions.

NRP-1 and OBR Complex Detection in MBA-MB231 Breast Cancer Cell Line byImmunocytochemistry Using a PLA Technology

The detection of NRP-1/OBR complex in human MDA-MB231 breast cancer cellline was assessed by proximity ligation assay (PLA) or duolinktechnology (www.olink.com). The detection of the NRP-1/OBR complex wasassessed on MDA-MB231 cell line cultivated in normal human serum (humanmale AB plasma, USA origin, MDL number MFCD00165829 H4522 Sigma) andtreated or not with Avastin 40 μg/ml final concentration for 48 h. Thegoal by using human serum was to mimic a physiologic condition duringtherapy with Avastin. The immunostained samples were analysed by theacquisition of the Z stacks through confocal microscopy on Zeiss LSM700, Inverted confocal microscope. The acquired images were analyzedusing Image J software for the quantification of NRP-1/OBR complexexpressed by cells.

Results

By using a BioLayer Interferometry technology (BLI,http://www.fortebio.com), we were able to demonstrate a directinteraction between recombinant proteins leptin and NRP-1 (FIG. 1A).VEGF165 has been used as positive control of the experiment.Surprisingly, in contrast to other NRP-1 ligands, VEGF, and Sema3A knownas competitors, leptin binds directly to NRP-1 but do not compete withVEGF binding and similarly VEGF does not prevent leptin binding toNRP-1. These observations suggest that leptin and VEGF should have adistinct binding domain.

Since BLI technology have shown that leptin and VEGF could interact withNRP-1 in non-competitive way and since we could demonstrate that leptinand VEGF form a complex in obese people tissue and by BLI technologyusing recombinant protein we assessed a docking of NRP-1 and leptincomplexed with VEGF165. From the best consensus mode, peptide sequencesSEQ ID NO:3 for leptin and SEQ ID NO:4 for NRP-1 have been identified(FIG. 2). The Leptin (OB) binding domain (SEQ ID:3) to NRP-1 (SEQ ID:2)was validated by BLI using a synthetized peptide (SEQ ID:3) and theextracellular domain of the recombinant protein NRP-1 from RnD systems(FIG. 3).

Since we have demonstrated that VEGF play a negative feed-backregulatory role for leptin signaling and since we demonstrated thatAvastin increased MDA-MB231 cell migration, this raises the question ofwhether of not the Avastin effect occurs or not through the increase ofNRP-1/OBR complex formation? Interestingly, compared to MDA-MB231 cellline cultivated in human serum, cells cultivated in the same conditionand treated with Avastin40 mg/ml presented a high number of NRP-1/OBRcomplex which my explain the increase of the migration of the cells.

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

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The invention claimed is:
 1. An isolated, a synthetic or a recombinantpolypeptide capable of inhibiting the binding between leptin and NRP-1,wherein the polypeptide consists of the amino acid sequence of SEQ IDNO:3 (ENLRDLLHVLAFSKSCHLPWASGLETL) or SEQ ID NO:4(EGNKPVLFOGNTNPTDVYVAVFPK), and wherein the polypeptide is fused to atleast one heterologous polypeptide.
 2. A nucleic acid encoding thepolypeptide fused to the at least one heterologous polypeptide ofclaim
 1. 3. The nucleic acid of claim 2 which is included in a suitablevector.
 4. An isolated host cell comprising the nucleic acid of claim 2.5. A pharmaceutical composition comprising the polypeptide fused to theat least one heterologous polypeptide of claim 1 or a nucleic acidencoding the polypeptide fused to the at least one heterologouspolypeptide.
 6. The nucleic acid of claim 3 wherein the vector is aplasmid, a cosmid, an episome, an artificial chromosome, a phage or aviral vector.